Modulation of regulatory T cells via G-coupled protein receptor 43

ABSTRACT

Disclosed herein are compositions and methods that are useful for inducing the development of regulatory T cells (Treg). Such compositions and methods are useful for treating inflammatory conditions and in particular inflammatory conditions affecting the gastrointestinal tract of a subject. In certain embodiments, the present inventions generally relate to short chain fatty acids and the discovery that such short chain fatty acids may be used to treat and/or prevent inflammatory conditions by enhancing the size and immune function of a subject&#39;s endogenous Treg population.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/777,476, filed Jan. 4, 2016, which is a national stage filing under35 U.S.C. 371 of International Application No. PCT/US2014/030832, filedMar. 17, 2014, which claims the benefit of U.S. Provisional ApplicationNo. 61/800,299, filed Mar. 15, 2013, the entire teachings of which areincorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with government support under grant numbersR01CA154426, F32DK095506 and K08AI078942 awarded by the NationalInstitutes of Health. The government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

Humans are colonized from birth with trillions of bacteria, the majorityof which reside within the intestinal tract and constitute the gutmicrobiota (Turnbaugh et al., Nature 449, 804-810 (2007)). Over themillennia the intestinal immune system has co-evolved with the gutmicrobiota to develop a number of highly regulated interactions requiredfor the maintenance of intestinal health. Disruption of this homeostasisleads to intestinal inflammation and is generally regarded as a rootcause of inflammatory bowel disease (Hooper, et al., Science 336,1268-1273 (2012)).

Regulatory T cells (T_(reg)) are a subpopulation of T cells that arecapable of modulating immune responses, and in particular modulatingautoimmune responses. Those T_(reg) expressing the transcription factorFoxp3 (i.e., Foxp3⁺ T_(reg)) are particularly important for limitingintestinal inflammation (Izcue, et al. Rev. 212, 256-271 (2006);Josefowicz, et al. Annu. Rev. Immunol. 30, 531-564 (2012)). Colonicregulatory T cells (cT_(reg)) represent a subpopulation of T_(reg) thatare active in the gastrointestinal tract and are critical for regulatingintestinal inflammation. Germ-free mice, which lack bacteria, havedecreased numbers of cT_(reg) and as a result are more susceptible tocertain experimental colitis models, therefore suggesting that cT_(reg)depend on signals derived from the microbiota for their properdevelopment and function (Geuking et al., Immunity 34, 794-806 (2011);Atarashi et al., Science 331, 337-341 (2011); Maslowski et al., Nature461, 1282-1286 (2009); Atarashi, et al., Semin. Immunol. 23, 146-153(2011)).

To date, it remains unclear how gut microbiota induce cT_(reg)responses, and the identification of compounds that promote adaptiveimmune maturation and influence intestinal T_(reg) and cT_(reg) remaincritical to understanding how intestinal homeostasis is maintained andhow it can be optimized to promote health. Additionally, novel therapiesare needed for modulating autoimmune and inflammatory responses, and inparticular autoimmune and immune responses affecting thegastrointestinal tract. Particularly needed are safe and effectivetherapies that are capable of inducing (e.g., enhancing) a subject'sability to mount an immune response. Also needed are safe and effectivetherapies that enhance the production and/or activation, or otherwiseincrease the quantity and/or function of, T_(reg) in thegastrointestinal tract of a subject.

SUMMARY OF THE INVENTION

The present inventors have discovered and disclose herein the mechanismsby which gut microbiota induce colonic regulatory T cell (cT_(reg))responses as well as compounds and agents that promote adaptive immunematuration and influence intestinal T_(reg). Also disclosed herein arenovel compositions and methods that are useful for treating or otherwisemodulating (e.g., reducing or otherwise improving) autoimmune andinflammatory responses and in particular modulating autoimmune andinflammatory responses involving the gastrointestinal tract (e.g.,treating colonic inflammation and inflammatory bowel diseases). Thecompositions and methods disclosed herein can be used to modulate (e.g.,increase, expand or otherwise improve) the quantity and/or the activityof colonic regulatory T cells (cT_(reg)), and thereby treat or preventdiseases or conditions having an inflammatory or autoimmune component.

In certain embodiments the inventions disclosed herein relate to methodsof increasing the quantity of cT_(reg), wherein such methods comprise astep of contacting a regulatory T cell (T_(reg)) with an effectiveamount of a composition to thereby increase the quantity of cT_(reg),and wherein the composition comprises one or more compounds capable ofmodulating (e.g., interacting with) a G-coupled protein receptor 43(GPR43). In some embodiments, the compounds comprise short chain fattyacids (e.g., one or more of formic acid, acetic acid, propionic acid,butyric acid, isobutyric acid, valeric acid, isovaleric acid andpharmaceutically acceptable salts, esters and pro-drugs thereof). Insome embodiments, the compounds are G-coupled protein receptor 43(GPR43) agonists.

Also provided herein are methods of increasing the function or activityof cT_(reg) (e.g., a Foxp3⁺ cT_(reg)) in the gastrointestinal tract of asubject, wherein the method comprises a step of contacting the cT_(reg)with a composition comprising one or more short chain fatty acids andthereby increasing cT_(reg) function or activity. For example, suchmethods may be employed to suppress an autoimmune response in a subject(e.g., a human subject with graft versus host disease).

In certain embodiments the inventions disclosed herein relate to methodsof increasing the suppressive capacity of cT_(reg), wherein such methodscomprise a step of contacting the cT_(reg) (e.g., a Foxp3⁺ and/or IL-10⁺cT_(reg)) with a composition comprising one or more short chain fattyacids and thereby increasing the suppressive capacity of the cT_(reg).

The inventions disclosed herein also relate to methods of increasing thequantity or concentration of cT_(reg), wherein such methods comprise astep of contacting a T_(reg) (e.g., Foxp3⁺/IL-10⁺ T_(reg)) with acomposition comprising one or more short chain fatty acids (e.g.,propionic acid and pharmaceutically acceptable salts, esters andpro-drugs thereof) and thereby increasing the quantity of the cT_(reg).

In still other embodiments, the inventions disclosed herein relate tomethods of modulating a cT_(reg) immune response in the gastrointestinaltract of a subject, wherein such methods comprise administering aneffective amount of a composition to the subject and thereby modulatingcT_(reg) immune response, and wherein the composition comprises one ormore short chain fatty acids (e.g., propionate). In some embodimentsmodulating cT_(reg) immune response comprises one or more of increasingthe quantity of cT_(reg) and increasing the function or activity (e.g.,the suppressive activity) of cT_(reg).

In certain embodiments, the inventions disclosed herein relate tomethods of treating or preventing colonic inflammation in a subject,wherein such methods comprise a step of administering an effectiveamount of a composition to the subject and thereby treating orpreventing colonic inflammation, wherein the composition comprises oneor more short chain fatty acids, and wherein the composition increasesimmune function of cT_(reg) in the gastrointestinal tract of thesubject.

Also disclosed are methods of reducing the incidence of relapse ofinflammatory bowel diseases (IBD) in a subject, wherein such methodscomprise administering an effective amount of a composition to thesubject and thereby reducing the incidence of relapse of IBD, whereinthe composition comprises one or more short chain fatty acids, andwherein the composition modulates the immune response of the subject'scT_(reg) (e.g., a Foxp3⁺/IL-10⁺ cT_(reg)). For example, theadministration of such compositions (e.g., compositions comprising oneor more short chain fatty acids) may cause an increase in the quantityor enhance the function of cT_(reg), thereby providing an improvedtherapeutic benefit and reducing the incidence of relapse of IBD.

In certain embodiments, also disclosed herein are methods of increasingthe expression of Foxp3 and IL-10 in Foxp3⁺ and IL-10⁺ T_(reg), whereinsuch methods comprise a step of contacting the T_(reg) with acomposition comprising one or more short chain fatty acids and therebyincreasing expression of Foxp3 and IL-10.

In some embodiments, the T_(reg) express Ffar2 or otherwise comprise aGPR43. In certain embodiments, the compositions comprise one or morecompounds or agents (e.g., short chain fatty acids) that modulate,interact with (e.g., bind to) or otherwise target the GPR43. Themodulation or interaction of such compounds or agents (e.g., short chainfatty acids) with the GPR43 inhibits histone deacetylase (HDAC). Forexample, in certain embodiments, the short chain fatty acids disclosedherein (e.g., propionate) may modulate or interact (e.g., bind to) oneor more GPR43 and thereby inhibit HDAC.

In certain embodiments, the modulation of the cT_(reg) (e.g., modulationof the cT_(reg) immune response in the gastrointestinal tract of amammalian subject) is mediated through HDAC inhibition. In someembodiments, the increase in the suppressive capacity of cT_(reg) ismediated through HDAC inhibition. In other embodiments, the observedincrease in the quantity of cT_(reg) is mediated through HDAC inhibitionby the one or more short chain fatty acids.

In certain embodiments disclosed herein, the T_(reg) or cT_(reg) isFoxp3⁺. In certain embodiments disclosed herein, the T_(reg) or cT_(reg)is IL-10⁺. In yet other embodiments, the T_(reg) or cT_(reg) is capableof expressing and/or producing IL-10.

The compounds and methods disclosed herein are particularly suitable fortreating inflammatory conditions (e.g., inflammatory bowel disease) andautoimmune conditions involving the gastrointestinal tract. In someembodiments, the inventions relate to methods of treating colonicinflammation, for example, treating a subject (e.g., a human subject)suffering from or otherwise affected by colonic inflammation that issecondary to an autoimmune disease (e.g., graft versus host disease). Incertain embodiments, the inventions relate to methods of treatinginflammation, for example, treating a subject (e.g., a mammal) havinginflammation that is secondary to inflammatory bowel disease (e.g.,Crohn's disease).

Also disclosed herein are compositions (e.g., pharmaceuticalcompositions) that comprise one or more agents or compounds that arecapable of modulating or otherwise interacting with GPR43. In certainembodiments, the compositions disclosed herein generally comprise one ormore short chain fatty acids. For example, the one or more short chainfatty acids may be selected from the group consisting of formic acid,acetic acid, propionic acid, butyric acid, isobutyric acid, valericacid, isovaleric acid and pharmaceutically acceptable salts, esters orpro-drugs thereof. In certain embodiments, the short chain fatty acidscomprise a mixture of one or more of formic acid, acetic acid, propionicacid, butyric acid, isobutyric acid, valeric acid, isovaleric acid andpharmaceutically acceptable salts, esters or pro-drugs thereof. Incertain embodiments, such short chain fatty acids increase the quantityof cT_(reg) (e.g., increase the concentration of cT_(reg) in the colonof a human subject).

Also contemplated are methods of contacting cT_(reg) ex vivo using thecompositions disclosed herein (e.g., compositions comprising one or moreshort chain fatty acids). In such embodiments such cT_(reg) may becontacted (e.g., contacted with one or more short chain fatty acids toincrease their quantity or function) and subsequently are administeredto a subject (e.g., to treat an inflammatory condition).

The invention also relates to methods of increasing expression of Foxp3and IL-10 in Foxp3⁺ and IL-10⁺ regulatory T cells (T_(reg)), wherein themethod comprises contacting the T_(reg) with a composition comprisingone or more short chain fatty acids (e.g., propionate) and therebyincreasing expression of Foxp3 and IL-10. In some embodiments, theT_(reg) are cT_(reg). In other embodiments, the T_(reg) express Ffar2 ora GPR43. In yet other embodiments, the interaction of the one or moreshort chain fatty acids with the GPR43 inhibits HDAC.

In certain embodiments, the methods and compositions disclosed hereinincrease the concentration of a GPR43 agonist in a subject (e.g., byincreasing the concentration of short chain fatty acids within the colonof a subject). For example, in certain embodiments, the administrationof short chain fatty acids (e.g., propionic acid, acetic acid and/orbutyric acid) to a subject may result in such short chain fatty acidconcentration of at least 5 μmol/g, 10 μmol/g, 15 μmol/g, 20 μmol/g, 25μmol/g, 30 μmol/g, 35 μmol/g, 40 μmol/g, 45 μmol/g, 50 μmol/g, 55μmol/g, 60 μmol/g, 65 μmol/g, 70 μmol/g, 75 μmol/g, 80 μmol/g, 85μmol/g, 90 μmol/g, 95 μmol/g, 100 μmol/g, 200 μmol/g, 250 μmol/g, 500μmol/g or more in the luminal contents of the subject. In certainembodiments, the concentration of propionate in the tissues of the colonis at least 0.1 mM. In certain embodiments, such concentrations of shortchain fatty acids (e.g., propionic acid, acetic acid and/or butyricacid) are achieved by administering an effective amount (e.g., at leastabout 0.1 mM, 0.5 mM, 1 mM, 2.5 mM, 5 mM, 10 mM, 50 mM, 75 mM, 100 mM,150 mM, 200 mM, 250 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM,900 mM, 1000 mM, or more) of the short chain fatty acids on at least adaily basis (e.g., at least once, twice, three times, four times, fivetimes, six times daily). In certain embodiments, an effective amount ofGPR43 agonist (e.g., short chain fatty acids) that is administered tothe subject is not achievable through dietary intake or dietarysupplementation.

The above discussed and many other features and attendant advantages ofthe present invention will become better understood by reference to thefollowing detailed description of the invention when taken inconjunction with the accompanying examples.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-1F illustrate that short chain fatty acids (SCFA) restorecolonic T_(reg) populations and function in germ-free (GF) andantibiotic treated mice. FIG. 1A depicts cecal SCFA levels that weremeasured from GF and specific pathogen-free (SPF) BALB/c WT mice byHPLC. Data represent the means and sum of 3 experiments with cecalcontents from 3-5 age and sex-matched mice per group. FIG. 1Billustrates colonic lamina propria (LP) lymphocytes that were isolatedand stained for CD4 and Foxp3. Upper panel: Representative flowgrams and% CD4⁺Foxp3⁺ within the CD45⁺CD3⁺ population from SPF, GF, and GF micetreated with propionic acid (P), acetic acid (A), butyric acid (B), orthe SCFA mix (MIX) in the drinking water. Lower panel: Numbers of Foxp3⁺T_(reg) for the upper panel. FIG. 1C illustrates that SCFA expandFoxp3⁺Helios⁺ T_(reg) in the colon of GF free mice. Colonic LPlymphocytes were isolated from SPF, GF, and GF mice treated with P, A, Bor the SCFA mix in the drinking water for three weeks and stained forCD45, CD4, Helios, and Foxp3. Upper panel: Representative flowgrams andpercentage of Foxp3⁺CD4⁺ population with Helios staining. Lower panel:Numbers of Helios⁺Foxp3⁺ T_(reg) for the upper panel. FIG. 1Dillustrates colonic T_(reg) (CT_(reg)) that were isolated from GF miceand purified by FACS staining for CD4, CD127, and CD25, cultured for 24hours in the presence or absence of 0.1 mM propionate, and examined forexpression of Foxp3, TGFβ and IL-10 mRNA by qPCR. FIG. 1E illustratescT_(reg) that were isolated from GF mice and purified as in FIG. 1D,cultured for 24 hours in the presence or absence of 0.1 mM propionateand examined for expression of Foxp3, TGFβ, and IL-10 by RTqPCR andIL-10 protein production by ELISA. FIG. 1F illustrates mice that weretreated orally with vancomycin for 2 weeks and then also given P, A, B,or the SCFA mix. After 4 weeks, colonic LP lymphocytes were isolated andstained for CD4 and Foxp3. The percentage of CD4⁺Foxp3⁺ within theCD45⁺CD3⁺ population from SPF mice or vancomyin-treated SPF mice exposedto water alone (−), P, A, B, or the SCFA mix are shown. For FIG. 1B,symbols represent data from individual mice. Horizontal lines show themean and error bars the SD. For FIGS. 1D and 1E, each symbol or barrepresents pooled cT_(reg) from 3-5 mice. All data shown arerepresentative of at least 3 independent experiments. A Kruskal-Wallistest with a Dunn's post hoc test was performed in FIGS. 1B and 1F, ***P<0.001 and * P<0.05. A Mann-Whitney U test was performed in FIGS. 1Dand 1E.

FIGS. 2A-2C illustrate that short chain fatty acids (SCFA) do not affectsplenic, mesenteric lymph node (MLN) or thymic Foxp3⁺ T_(reg)populations in germ-free (GF) mice. Lymphocytes were isolated from thespleen (FIG. 2A), MLN (FIG. 2B) or thymus (FIG. 2C) and stained for CD4and Foxp3. Upper panels: Representative flowgrams. Lower panels:Percentage of and number of CD4⁺Foxp3⁺ within the CD45⁺CD3⁺ populationfrom SPF, GF, and GF mice treated with propionic acid (P), acetic acid(A) or butyric acid (B) in the drinking water per mouse colon, asindicated. Each symbol represents data from an individual mouse and datareflect 3-5 independent experiments. P-values are not shown asdifferences with SCFA treatment were not statistically significant.Horizontal lines show the mean and error bars the SD.

FIG. 3 shows that short chain fatty acids (SCFA) affect total colonicCD4⁺ T cells. Lymphocytes were isolated from the colon and stained forCD4 and Foxp3. Upper panel: Percentage of, and Lower panel: Number of,CD4⁺ within the CD45⁺CD3⁺ population from SPF, GF, and GF mice treatedwith propionic acid (P), acetic acid (A), butyric acid (B), or SCFA mixper mouse colon. Each symbol represents data from an individual mouseand the data reflect 3-5 independent experiments. P-values are not shownas differences with SCFA treatment were not statistically significant.Horizontal lines show the mean and error bars the SD.

FIGS. 4A-4D illustrate that short chain fatty acids (SCFA) do not affectcolonic Th1 and Th17 cells in germ-free (GF) mice. Lymphocytes wereisolated from the colon of SPF, GF and GF mice treated with propionicacid (P) or acetic acid (A) in the drinking water for three weeks andstained for CD45, CD3, CD4, IFNγ, and IL-17. FIG. 4A depictsrepresentative flowgrams of CD45⁺CD3⁺CD4⁺IFNγ⁺ populations. FIG. 4B inthe upper right panel: Percentage of, and Lower right panel: Number of,CD4⁺IFNγ⁺ cells within the CD45⁺CD3⁺ population. FIG. 4C depictsrepresentative flowgrams of CD45⁺CD3⁺CD4⁺IL-17⁺ populations. FIG. 4D inthe Upper right panel: Percentage of, and Lower right panel: Number of,CD4⁺IL-17⁺ cells within the CD45⁺CD3⁺ population. Each symbol representsdata from an individual mouse and data reflect 3 independentexperiments. P-values are not shown as differences with SCFA treatmentwere not statistically significant. Horizontal lines show the mean anderror bars the SD.

FIGS. 5A-5F illustrate that short chain fatty acids (SCFA) augmentcolonic T_(reg) (cT_(reg)) population size and function in SPC mice.FIG. 5A illustrates the results of SPF Foxp3^(YFP-Cre) mice that weretreated with water alone (−) or propionic acid (P), acetic acid (A) orbutyric acid (B), or the SCFA mix. Colonic lamina propria (LP)lymphocytes were isolated and stained for CD4 and IL-10. Upper panel:Representative dot plots and percentage of CD4⁺ Foxp3-YFP⁺ within theCD45⁺CD3⁺ population. Lower panel: Representative dot plots andpercentage of the CD4+Foxp3+IL-10+population. FIG. 5B corresponds to thecell numbers for the data in FIG. 5A upper panel. FIG. 5C corresponds tothe cell numbers for the data in FIG. 5A lower panel. Symbols in FIGS.5B and 5C represent data from individual mice and represent 4independent experiments. FIG. 5D illustrates that SCFA increase colonicT_(reg) suppressive capacity. Colonic LP T_(reg) and splenic effector Tcells were isolated by FACS sorting. Splenic effector T cells were firstlabeled with CellTrace cell violet dye (5 μM) and cultured alone orco-cultured with cT_(reg). Cultures were either unstimulated orstimulated with 1 μg/ml plate bound anti-CD3, 1 μg/ml soluble CD28 and Por pH and sodium matched media for 96 hrs. Flowgrams depicted arerepresentative of three independent experiments. Significance is shownin FIG. 5E. Horizontal lines represent the mean and error bars the SD.FIG. 5E depicts the suppression of cT_(reg) that were co-cultured withsplenic effector T cells (Teff) and P, A, B, or media (sodium and pHmatched) for 96 hours. Percent suppression (y-axis), Treg:Teff ratios(x-axis). Symbols represent mean values and error bars SD for fourindependent experiments. * P<0.01. FIG. 5F illustrates that in vitrotreatment of colonic T_(reg) with propionate increases Foxp3 and IL-10expression, as well as IL-10 protein production. Colonic LP T_(reg) wereisolated from SPF Foxp3^(YFP-Cre) mice, cultured for 24 hours in thepresence or absence of 0.1 mM propionate, and examined for in vitroexpression of Foxp3, and IL-10 by RTqPCR or secretion of IL-10 by ELISA.Upper left panel: expression of Foxp3. Upper middle panel: expression ofTGFβ. Lower middle panel: TGFβ protein production. Upper right panel:expression of IL-10. Lower right panel: IL-10 protein production. Eachsymbol represents pooled colonic T_(reg) from 3-4 mice and reflects datafrom at least 4 independent experiments. Mann-Whitney U test wasperformed to determine statistical significance. Horizontal linesrepresent the mean and error bars the SD.

FIG. 6 illustrates the relative expression of Foxp3 and IL-10 fromcT_(reg) isolated from the lamina propria (LP) of in vivo propionatetreated SPF Foxp3^(YFP-Cre) mice, sorted for CD4 and YFP. Each symbolrepresents pooled cT_(reg) from 3-5 mice, horizontal lines show the meanand error bars the SD. Four independent experiments were performed. AMann-Whitney U test was performed and P-values are shown.

FIG. 7 shows that treatment with propionate enhances proliferation ofcolonic T_(reg) (cT_(reg)). CD4⁺ Foxp3-YFP⁺ colonic T_(reg) were FACSsorted, labeled with CellTrace cell violet (5 μM) and cultured with 1μg/ml anti-CD3 and 500 ng/ml IL-2 in the presence or absence of 0.1 mMpropionate. Histograms showing cell division are shown in the upperpanel with average percentage of divided population ±SD on thehistogram. Division index is plotted for the experimental conditions inthe lower panel. Data are representative of two independent experiments.Student's t-test was performed to determine statistical significance.Horizontal lines represent the mean and error bars the SD.

FIG. 8 presents chemokine receptor expression in colonic T_(reg) fromwater vs. propionate-treated germ-free (GF) and specific pathogen-free(SPF) mice. Colonic lamina propria (LP) T_(reg) were isolated from wateror in vivo propionate-treated Swiss Webster GF (left panels) or SPFFoxp3^(YFP-Cre) (right panels) mice and the ex vivo expression of thechemokine receptors CCR9, α4 integrin, β7 integrin and GPR15 weremeasured by RTqPCR. Data consist of 5 pooled mice per group and arerepresentative of two independent experiments. Student's t-test wasperformed to determine significance. Error bars represent SD.

FIG. 9 illustrates that short chain fatty acids (SCFA) do not affectTGFβ levels of colonic Foxp3⁺ T_(reg) populations in SPF mice. BALB/cSPF mice were treated with pH and sodium-matched water alone (−),propionic acid (P), acetic acid (A) or butyric acid (B), or the SCFA mixin the drinking water for two weeks. Colonic lamina propria (LP)lymphocytes were isolated and stained for CD45, CD4, Foxp3, and TGF-β.Upper panel: Representative flowgrams. Lower panel: Percentage ofCD4⁺Foxp3⁺ T_(reg) expressing TGFβ within the CD45⁺ population from allof the treatment groups. Each symbol represents data from an individualmouse and the data reflect 3 independent experiments. P-values are notshown as differences with SCFA treatment were not statisticallysignificant. Horizontal lines represent the mean and error bars the SD.

FIG. 10 illustrates that propionate does not affect small intestinalFoxp3⁺ T_(reg) populations in SPF mice. Lamina propria (LP) lymphocyteswere isolated from the distal 10 cm of the ileum and stained for CD45,CD4, and Foxp3. Left panel: Representative flowgrams. Middle panel:Percentage of, and Right panel: Number of, CD4⁺Foxp3⁺ within the CD45⁺population from SPF mice treated with propionate or pH andsodium-matched water per mouse distal ileum. Each symbol represents datafrom an individual mouse and data reflect two independent experiments.P-values are not shown as differences with SCFA treatment were notstatistically significant. Horizontal lines represent the mean and errorbars the SD.

FIG. 11 shows that short chain fatty acids (SCFA) do not affect colonicTh17 populations in specific pathogen-free (SPF) mice. BALB/c SPF micewere treated with pH and sodium-matched water alone (−), propionic acid(P), acetic acid (A) or butyric acid (B), or the SCFA mix. Coloniclamina propria (LP) lymphocytes were isolated and stained for CD45, CD4,Foxp3, and IL-17. Upper panel: Representative flowgrams. Lower leftpanel: Percentage of Foxp3-CD4⁺ T cells expressing IL-17 per mousecolon. Lower right panel: Number of Foxp3-CD4⁺ T cells expressing IL-17per mouse colon. Symbols represent data from individual mice. Datareflect three-five independent experiments. P-values are not shown asdifferences with SCFA treatment were not statistically significant.Horizontal lines represent the mean and error bars the SD.

FIG. 12 illustrates that short chain fatty acids (SCFA) do not affectcolonic Th1 populations in specific pathogen-free (SPF) mice. BALB/c SPFmice were treated with pH and sodium-matched water alone (−), propionicacid (P), acetic acid (A) or butyric acid (B), or the SCFA mix. Coloniclamina propria (LP) lymphocytes were isolated and stained for CD45, CD4,and IFNγ. Upper panel: Representative flowgrams. Lower panel: Percentageof CD4⁺ T cells expressing IFNγ within the CD45⁺ population from all ofthe treatment groups. Symbols represent data from individual mice andreflect data from three independent experiments. P-values are not shownas differences with SCFA treatment were not statistically significant.Horizontal lines represent the mean and error bars the SD.

FIG. 13 illustrates that short chain fatty acids (SCFA) do not affectmesenteric lymph node (MLN) Foxp3⁺ T_(reg) populations in specificpathogen-free (SPF) mice. Lymphocytes were isolated from the MLNs ofBALB/c SPF mice treated with pH and sodium-matched water alone (−),propionic acid (P), acetic acid (A) or butyric acid (B), or the SCFA mixand stained for CD45, CD4, and Foxp3. Upper panel: Representativeflowgrams. Lower Left panel: Percentage of, and Lower right panel:Number of, CD4⁺Foxp3⁺ within the CD45⁺ population. Symbols representdata from individual mice and reflect data from three independentexperiments. P-values are not shown as differences with SCFA treatmentwere not statistically significant. Horizontal lines represent the meanand error bars the SD.

FIG. 14 illustrates that short chain fatty acids (SCFA) do not affectsplenic Foxp3⁺ T_(reg) populations in specific pathogen-free (SPF) mice.Lymphocytes were isolated from the spleen of BALB/c SPF mice treatedwith pH and sodium-matched water alone (−), propionate (P), or the SCFAmix (Mix) and stained for CD45, CD4, and Foxp3. Upper panel:Representative flowgrams. Lower panel: Percentage of CD4⁺Foxp3⁺ withinthe CD45⁺ population. Symbols represent data from individual mice andreflect data from two independent experiments. P-values are not shown asdifferences with SCFA treatment were not statistically significant.Horizontal lines represent the mean and error bars the SD.

FIGS. 15A-15I show that Ffar2 mediates SCFA effects on colonic T_(reg)(dT_(reg)). FIG. 15A illustrates T_(reg) that were isolated from thecolon, small intestine, spleen and mesenteric lymph node (MLN) ofspecific pathogen-free (SPF) and germ-free (GF) BALB/c mice, purified asdescribed in FIG. 1D and Ffar2 expression examined by qPCR. Each symbolrepresents data from 3-5 individual mice, horizontal lines show the meanand error bars the SD. Data reflect 3-7 independent experiments. FIG.15B shows results of lymphocytes that were isolated from the colon,small intestine, spleen, and MLN of SPF Ffar2^(−/−) and littermateFfar2^(+/+) mice. Cells were stained for CD4, Foxp3, and Ffar2. Leftpanel depicts a representative flow cytometry histogram comparingcolonic Ffar2 expression in Ffar2^(−/−) vs littermate Ffar2^(+/+) mice.Right panel shows the mean fluorescent intensity (MFI) for Ffar2 forT_(reg) from the indicated sites. Bars show the mean, error bars SD, anddata reflect 4 independent experiments. FIG. 15C illustrates that Ffar2receptor (GPR43) is highly expressed in colonic T_(reg) and myeloidcells. Colonic lamina propria (LP) lymphocytes were isolated from SPFFfar2^(+/+) and Ffar2^(−/−) mice, stained for CD45, CD4, Foxp3 and CD11band the MFI was determined by flow cytometry. The MFI was calculated byaveraging the geometric means for Ffar2 expression of individual mice.The bar graph shown is the average of 5 individual mice per group and isrepresentative of 3 independent experiments. Horizontal lines representthe mean and error bars the SD. FIG. 15D depicts results of colonic LPlymphocytes that were isolated from Ffar2^(−/−) and littermateFfar2^(+/+) mice exposed to propionate (P) or water alone and stainedfor CD4 and Foxp3. Left panel: Representative dot plots with percentageof CD4⁺Foxp3⁺ within the CD45⁺CD3⁺ population. Right panel: Foxp3⁺T_(reg) number for the left panel. Each symbol represents data fromindividual mice, horizontal lines show the mean and error bars the SD.FIG. 15E illustrates that Ffar2 is required for propionate-mediatedincrease of Foxp3 and IL-10 levels in colonic T_(reg) from SPF mice.Colonic LP T_(reg) were isolated from Ffar2^(−/−) and littermateFfar2^(+/+) mice, purified as described in FIG. 1D, cultured in thepresence of 0.1 mM propionate for 24 hours and examined for expressionof Foxp3 and IL-10 by RTqFCR and secretion of IL-10 by ELISA. Eachsymbol represents pooled data from 3-5 mice and data are representativeof 2 independent experiments. Student's t-test was performed todetermine significance. Horizontal lines represent the mean and errorbars the SD. FIG. 15F shows that Ffar2 is required for thepropionate-mediated restoration of colonic T_(reg) levels in SPFantibiotic treated mice. Ffar2^(−/−) and littermate Ffar2^(+/+) micewere treated orally with vancomycin for 4 weeks and also givenpropionate (P) in the drinking water starting in week 2. After 4 weeks,colon LP lymphocytes were isolated and stained for CD45, CD4 and Foxp3.The percentage and number of CD4⁺Foxp3⁺ cells within the CD45+population are shown. Each symbol represents data from an individualmouse and data represent 2 independent experiments. Student's t-test wasperformed to determine significance. Horizontal lines represent the meanand error bars the SD. FIG. 15G shows the results of cT_(reg) that wereisolated from the LP of Ffar2^(−/−) and littermate Ffar2^(+/+) mice,purified as described in FIG. 1D, cultured in the presence of 0.1 mMpropionate or media (pH and sodium matched) for 24 hours and examinedfor expression of HDAC 1, 2, 6, 7 and 9 by RTqFCR. Bars show the meanand error bars the SD of 3 independent experiments. FIG. 15H depictsresults of Ffar2^(−/−) and littermate Ffar2^(+/+) cT_(reg) that wereco-cultured with splenic T_(eff) cells in media with or withoutpropionate for 96 hours. Percent suppression (y-axis) andT_(reg):T_(eff) ratios (x-axis). Symbols represent the mean of 3independent experiments and error bars show the SD. FIG. 15I present theresults of whole cell extracts that were generated from cT_(reg)isolated from the LP of Ffar2^(−/−) and littermate Ffar2^(+/+) mice,purified as described in FIG. 1D, and cultured in the presence of 0.1 mMpropionate or media (pH and sodium matched) for 24 hours. Samples wereanalyzed by Western blotting for histone acetylation by examining levelsof acetylated histone (H3K9), total histone levels were used as aloading control. The Western blot shown is representative of twoindependent experiments with cT_(reg) cell lysates pooled from 10-12mice per group. A bar graph of densitometry ratios of acetylated HistoneH3:total Histone H3 is shown. Bars represent the mean and error bars theSD. A Kruskal-Wallis test with a Dunn's post hoc test was performed forFIGS. 15A and 15H, *** P<0.001. The Mann-Whitney U test was performedfor FIGS. 15D and 15G. The student's t-test was performed for FIGS. 15Band 15I.

FIGS. 16A and 16B illustrate that SCFA treatment reduced colitisseverity and pro-inflammatory cytokine expression and increased IL-10expression during T cell transfer colitis. BALB/c Rag2^(−/−) mice wereinjected with CD4⁺CD45RB^(hi)CD25⁻ naïve T cells alone or in combinationwith T_(reg). Following injection, mice received propionate, SCFA mix,or pH and sodium-matched drinking water. FIG. 16A shows the histologiccolitis score along the y-axis, the treatment groups and experimentalconditions are shown along the x-axis. FIG. 16B illustratesrepresentative H&E images for the experimental groups. Propionate andSCFA treated mice show reduced degrees of colonic crypt injury,inflammation, and hyperplasia compared to control mice. A 100 μm scalebar is shown in the lower left of each image. One-way ANOVA withBonferroni post-hoc is shown in FIG. 16A. Horizontal lines represent themean and error bars the SD.

FIGS. 17A-17F illustrate that SCFA exposure ameliorates T cell transfercolitis in a T_(reg)-intrinsic, Ffar2-dependent manner. BALB/cRag2^(−/−) mice were injected with CD4⁺CD45RB^(hi)CD25^(lo) naïve Tcells alone or in combination with T_(reg). Following injection, micereceived propionate, SCFA mix, or pH and sodium-matched drinking water.FIG. 17A depicts the weekly percentage body weight change across theexperimental groups from experimental day 0 through day 42. Symbols showthe mean and error bars the SD. Data reflect three independentexperiments. Colonic lamina propria (LP) lymphocytes were isolated andstained for CD4 and Foxp3 and percentage and number of CD4⁺Foxp3⁺ withinthe CD45⁺CD3⁺ population are shown in FIGS. 17B and 17C, respectively.Symbols represent data from individual mice, horizontal lines show themean and error bars the SD. FIGS. 17D-F show the results of C57BL/6Rag2^(−/−) mice that were injected with CD4⁺CD45RB^(hi)CD25^(lo) naïve Tcells alone or in combination with Ffar2^(+/+) or Ffar2^(−/−) T_(reg).Following injection mice received propionate or pH and sodium-matcheddrinking water. In FIG. 17D, histologic colitis score is shown along they-axis, and the treatment group and experimental conditions are shownalong the x-axis. Colonic LP lymphocytes were isolated and percentageand number of CD4⁺Foxp3⁺ within the CD45⁺CD3⁺ population are shown inFIGS. 17E and 17F, respectively. Symbols represent data from individualmice. Horizontal lines show the mean and error bars the SD. FIGS.17D-17F represent data from 2 independent experiments. TheKruskal-Wallis test with a Dunn's post hoc test was performed for FIGS.17A-17F. ** P<0.01, * P<0.05.

FIG. 18 shows that Ffar2 is required for SCFA-mediated protectionagainst weight loss during T cell mediated colitis. C57BL/6 RAG2^(−/−)mice were injected with CD4⁺CD45RB^(hi)CD25⁺ naïve T cells alone or incombination with Ffar2^(+/+) or Ffar2^(−/−) T_(reg). Following injectionmice received propionate or pH and sodium-matched drinking water. Weeklypercentage body weight change is shown across the experimental groupsfrom experimental day 0 through day 63. Each data point is the averageof 5-7 individual mice and data are representative of 2 independentexperiments. Students' t-test was performed to determine significance,** indicates P<0.01, * P<0.05. Horizontal lines represent the mean anderror bars the SD.

FIG. 19 illustrates that propionate in the drinking water andintrarectal instillation of propionate affect similar changes in colonicT_(reg) populations in SPF mice. Colonic lamina propria (LP) Lymphocyteswere isolated and stained for CD45, CD4, and Foxp3 from mice treatedwith propionate in the drinking water or treated with propionateintrarectally for two weeks. The frequency of CD4⁺Foxp3⁺ within theCD45⁺ population from SPF mice treated with pH- and sodium-matched wateralone or propionate is shown. Each symbol represents data fromindividual mice and show data from two independent experiments.Horizontal lines represent the mean and error bars the SD.

FIG. 20 depicts Ffar2 expression patterns. Upper panels: Colonic laminapropria (LP) lymphocytes were isolated from Ffar2^(+/−) and Ffar2^(+/+)littermates, purified by FACS staining for CD4, CD127, and CD25, andexamined ex vivo for expression of Ffar2 and Foxp3 by RTqFCR. Eachsymbol represents data from 5-8 mice and data reflect three independentexperiments. Lower panel: Ffar2 expression levels were examined inFoxp3-YFP⁻ and Foxp3-YFP⁺ CD4+ T cells isolated from the colon, smallintestine (SI), mesenteric lymph node (MLN), and spleen by RTqPCR.Horizontal lines represent the mean and error bars the SD.

DETAILED DESCRIPTION OF THE INVENTION

The inventions described herein generally relate to the findings thatshort chain fatty acids (SCFA) play a critical role in the induction ofregulatory T cells (T_(reg)), and in particular colonic regulatory Tcells (cT_(reg)). T_(reg) are a subpopulation of T cells that arecapable of modulating immune responses and those T_(reg) expressing thetranscription factor Foxp3 (i.e., Foxp3⁺ T_(reg)) are particularlyimportant for limiting intestinal inflammation (Izcue, et al. Rev. 212,256-271 (2006); Josefowicz, et al. Annu. Rev. Immunol. 30, 531-564(2012)). cT_(reg) are a subpopulation of T_(reg) that are active in thegastrointestinal tract and are involved in maintaining colonichomeostasis and limiting inflammation of the gastrointestinal tract.

Disclosed herein are the mechanisms pursuant to which SCFA mediatecT_(reg) responses, and thereby mitigate intestinal inflammatoryresponses (e.g., inflammatory responses related to inflammatory boweldiseases). Also disclosed are novel compositions and optimized methodsof maintaining intestinal homeostasis and of treating or preventingcolonic inflammation.

The methods and compositions disclosed herein are useful for thetreatment of inflammatory conditions, and in particular colonicinflammation. As is used herein, the phrase “colonic inflammation”broadly refers to any inflammatory disease, disorder or pathologicalcondition affecting the cells and tissues of the gastrointestinal tract.Exemplary involving colonic inflammatory conditions include inflammatorybowel disease, pouchitis, aging, irritable bowel syndrome, and cancer ofthe gastrointestinal tract, obesity and type II diabetes. In certainembodiments, the colonic inflammation is secondary to an immune disorderor an autoimmune disorder. For example, the compositions and methodsdisclosed herein may be employed to treat or otherwise reduce colonicinflammation which occurs as a result of, or is otherwise associatedwith graft versus host disease.

In certain embodiments, the methods and compositions disclosed hereinmay be employed to treat or prevent relapses of inflammatory boweldisease (IBD). For example, disclosed herein are methods of reducing theincidence of relapse of IBD in a subject, by administering an effectiveamount of a composition comprising one or more short chain fatty acidsto a subject. As used herein, the phrases “inflammatory bowel disease”and “IBD” broadly refer to a set of chronic, idiopathic, immune-mediateddisorders that result in the inflammation of the gastrointestinal tract,and such phrases collectively include each of ulcerative colitis,Crohn's disease, irritable bowel syndrome, cancers of thegastrointestinal tract and pouchitis.

The present inventions are generally based upon the findings that shortchain fatty acids (SCFA) are capable of mediating the T_(reg) immuneresponse (e.g., the cT_(reg) immune response) by increasing or otherwisestimulating T_(reg) numbers and functional capacity and therebymodulating (e.g., increasing or otherwise enhancing) a subject's (e.g.,a human subject's) immune response. Such findings therefore providemeans of influencing the degree to which T_(reg) participate in aninflammation- or immune-mediated condition (e.g., colonic inflammationand/or IBD) and provide means of restoring intestinal homeostasis tothereby treat colonic inflammation.

As used herein, the phrases “short chain fatty acids” and “SCFA”generally refer to fatty acids having less than about ten carbons in thecarbon backbone and salts, esters and pro-drugs thereof. Exemplary SCFAinclude formic acid, acetic acid, propionic acid, butyric acid,isobutyric acid, valeric acid, isovaleric acid and pharmaceuticallyacceptable salts thereof. In certain embodiments, the short chain fattyacids comprise a mixture of one or more of formic acid, acetic acid,propionic acid, butyric acid, isobutyric acid, valeric acid, isovalericacid and pharmaceutically acceptable salts, esters or pro-drugs thereof.In certain embodiments, the short chain fatty acids comprise a mixtureof propionic acid, acetic acid and butyric acid.

The present inventions are also based on the findings that SCFA mediatethe induction of T_(reg), and in particular cT_(reg), by way of theG-coupled protein receptor 43 (GPR43) and that GPR43 is necessary forSCFA-mediated cT_(reg) induction. As discussed in greater detail below,GPR43 is encoded by the Ffar20 gene and is required for completeinduction of cT_(reg) by SCFA. It should be noted that while certainaspects of the present inventions contemplate the induction of cT_(reg)using SCFA, such inventions are not limited to SCFA. Also disclosed arecompositions and related methods of inducing (e.g., increasing thenumber and/or function) cT_(reg) by contacting a T_(reg) or a cT_(reg)with one or more compounds capable of modulating (e.g., interacting withor binding to) a GPR43 receptor.

In certain embodiments, the compositions and methods disclosed hereincause an increase in the quantity of cT_(reg). Similarly, in someembodiments, the compositions and methods disclosed herein augment(e.g., enhance, promote or increase) the native or natural function(e.g., the suppressive capacity) of cT_(reg). By increasing the quantityand/or function of cT_(reg), such cT_(reg) are able to exert suppressiveproperties in the gastrointestinal tract to a greater degree. As usedherein to describe T_(reg), the phrase “suppressive capacity” generallyrefers to the functional ability of T_(reg) to suppress inflammatory orimmune-mediated responses. In certain embodiments, the suppressivecapacity of cT_(reg) is a function of the quantity of cT_(reg) availableto exert their effects in the gastrointestinal tract of a subject. Inother embodiments, the suppressive capacity of cT_(reg) relates to thefunctional ability of the cT_(reg) in the gastrointestinal tract of asubject. The ability of the SCFA disclosed herein to augment, increaseor otherwise enhance T_(reg) function and quantity, represents aclinically significant advancement and can be applied to restoreintestinal homeostasis and treat, prevent or otherwise ameliorateintestinal or colonic inflammation.

The present inventors have also discovered that the GPR43-mediatedincreases in the quantity and suppressive capacity of cT_(reg) occursthrough histone deacetylase (HDAC) inhibition. In certain embodiments,the SCFA disclosed herein (e.g., propionate) increase the quantity andsuppressive capacity of cT_(reg) by reducing cT_(reg) expression ofHDAC6 (Class IIB) and/or HDAC9 (Class IIA).

In certain embodiments, the T_(reg) or cT_(reg) that is contacted or theactivity or quantity of which is otherwise modulated expresses Foxp3 oris Foxp3⁺. In certain embodiments, such T_(reg) or cT_(reg) expressedIL-10 or is IL-10⁺. In yet other embodiments, such T_(reg) or cT_(reg)is capable of expressing and/or producing IL-10.

In certain aspects, the methods of the present invention comprise theadministration of an effective amount of one or more SCFA (e.g., aceticacid and pharmaceutically acceptable salts thereof) to a subject havingcolonic inflammation (e.g., an immune-mediated disease or ulcerativecolitis). As used herein, the term “subject” means any mammal, includinghumans. In certain embodiments of the present invention the subject isan adult or an adolescent.

In certain embodiments, the inventions disclosed herein relate tomethods of contacting cT_(reg) ex vivo using the compositions disclosedherein (e.g., compositions comprising one or more short chain fattyacids). As the term is used herein, “contacting” generally refers tobringing two or more molecules or entities into close proximity witheach other such that such molecules or entities can react or otherwiseinteract with each other. A step of contacting may be performed invitro, ex vivo, in vivo. For example, in some embodiments a cT_(reg) maybe contacted with one or more short chain fatty acids (e.g., to increasethe quantity or function of such cT_(reg)) and subsequently areadministered to a subject (e.g., to treat an inflammatory condition).

As used herein, the phrase “effective amount” means an amount sufficientto achieve a meaningful benefit (e.g., improving or otherwiseameliorating colonic inflammation and/or maintaining intestinalhomeostasis). An effective amount of the SCFA (e.g., butyric acid) inthe compositions of the present invention may be generally determinedbased on the ability of such SCFA to stimulate or promote the quantityor function of cT_(reg). Generally, the amount of SCFA administered to asubject will depend upon the characteristics of the subject and theseverity of the subject's colonic inflammation. In certain embodiments,the compositions may be administered to a subject (e.g., administeredorally) once daily, twice daily, three times daily, four times daily,five times daily, six times daily, seven times daily, eight times daily,or more.

The compositions of the present invention can be administered to asubject by any suitable routes of administration. Preferably, followingthe administration of such compositions a therapeutic concentration ofthe SCFA (e.g., acetate) is achieved and/or maintained in the tissues ofthe gastrointestinal tract (e.g., within the lumen of thegastrointestinal tract). For example, in certain embodiments, thecompositions achieve and/or maintain a concentration of at least 5 mM,10 mM, 25 mM, 40 mM, 50 mM, 60 mM, 75 mM, 100 mM, 125 mM, 150 mM, 175mM, 200 mM, 250 mM, 300 mM, 400 mM, 500 mM or more within the lumen ofthe gastrointestinal tract. In certain embodiments, the composition iscombined with suitable excipients and formulated for enteral or rectaladministration. Alternatively, in certain embodiments, the compositionsof the present invention may be prepared for parenteral administration.General techniques applicable to the formulation and administration ofthe compositions of the present invention may be found in “Remington'sPharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latestedition.

The compositions of the present invention can also be administered orco-administered as part of a therapeutic regimen with other suitabletherapeutic or prophylactic agents (e.g., administered concurrently orsequentially). For example, the compositions disclosed herein may beformulated or co-administered with one or more additional therapeuticagents (e.g., one or more non-steroidal anti-inflammatory drugs,corticosteroids, chemotherapeutics, immunosuppressants and antibiotics).

In embodiments where the compositions are administered to a subjectorally, such compositions may be prepared or formulated as a dietarysupplement or as a functional or medical food. In other embodiments,such compositions may be prepared or formulated, for example, as apharmaceutical.

The articles “a” and “an” as used herein in the specification and in theclaims, unless clearly indicated to the contrary, should be understoodto include the plural referents. Claims or descriptions that include“or” between one or more members of a group are considered satisfied ifone, more than one, or all of the group members are present in, employedin, or otherwise relevant to a given product or process unless indicatedto the contrary or otherwise evident from the context. The inventionincludes embodiments in which exactly one member of the group is presentin, employed in, or otherwise relevant to a given product or process.The invention also includes embodiments in which more than one, or theentire group members are present in, employed in or otherwise relevantto a given product or process. Furthermore, it is to be understood thatthe invention encompasses all variations, combinations, and permutationsin which one or more limitations, elements, clauses, descriptive terms,etc., from one or more of the listed claims is introduced into anotherclaim dependent on the same base claim (or, as relevant, any otherclaim) unless otherwise indicated or unless it would be evident to oneof ordinary skill in the art that a contradiction or inconsistency wouldarise. Where elements are presented as lists, (e.g., in Markush group orsimilar format) it is to be understood that each subgroup of theelements is also disclosed, and any element(s) can be removed from thegroup. It should be understood that, in general, where the invention, oraspects of the invention, is/are referred to as comprising particularelements, features, etc., certain embodiments of the invention oraspects of the invention consist, or consist essentially of, suchelements, features, etc. For purposes of simplicity those embodimentshave not in every case been specifically set forth in so many wordsherein. It should also be understood that any embodiment or aspect ofthe invention can be explicitly excluded from the claims, regardless ofwhether the specific exclusion is recited in the specification. Theentire contents of all of the references (including literaturereferences, issued patents and published patent applications andwebsites) cited throughout this application are hereby expresslyincorporated by reference.

EXAMPLES Example 1

Humans and mice rely on bacteria to breakdown un-digestible materialsingested as part of their diets, such as fibers and other complexpolysaccharides. The main end products of this bacterial fermentationare short chain fatty acids (SCFA). In the present study, concentrationsof SCFA in specific pathogen-free (SPF) mice, gnotobiotic AlteredSchaedler Flora (ASF)-colonized mice, and germ-free (GF) mice wereevaluated and, as depicted in FIG. 1A, GF mice had reducedconcentrations of the three most abundant luminal SCFA, acetic acid,propionic acid and butyric acid (see, Table 1 below). This substantialdecrease of the three most abundant luminal SCFA in GF mice suggeststhat SCFA may contribute to some of the immune defects observed in GFmice and in particular the reduced number of cT_(reg).

The present investigators provided SCFA in the drinking water (150 mM)of GF mice for three weeks and, as depicted in FIG. 1B, observed thateach SCFA individually or in combination (SCFA mix) significantlyincreased the percentage and number of cT_(reg). The observed effectswere restricted to the colon, as T_(reg) numbers and frequency in thespleen, mesenteric lymph nodes (MLNs) and thymus were unchanged (FIGS.2A, 2B and 2C). These effects coincided with increased luminal SCFA(Table 1). SCFA increased CD4+ T cell frequency and number (FIG. 3), butdid not alter colonic Th1 or Th17 cell numbers significantly (FIG. 4).

Microbiota-induced cT_(reg) development is associated with an increasein de novo generation of inducible T_(reg) (iT_(reg)) and not in T_(reg)of thymic origin (nT_(reg)) (Atarashi, et al., Science 331, 337-341(2011)). These two populations can be distinguished by their expressionof the transcription factor Helios, which in vivo is restricted tonT_(reg) (Thornton et al., J. Immunol. 184, 3433-3441 (2010)). Asillustrated in FIG. 1C, the present investigators determined that theproportion of Helios⁺ T_(reg) remained unchanged between GF mice and GFmice treated with SCFA and was significantly greater than in SPF mice,indicating an expansion in T_(reg) already present in the colonic laminapropria (cLP) rather than de novo generation.

TABLE 1 SCFA levels Cecal or Si contents SPF ASF GF GF + P GF + A GF + BGF + Mic SPF + P μmol/g luminal contente Propionate  21.90 ± 0.122 18.83± 2.72 1.46 ± 0.162 14.93 ± 4.50 — — 11.78 ± 6.38 43.33 ± 12.17 Acetate40.66 ± 5.86 28.69 ± 8.32 2.82 ± 0.534 — 16.18 ± 4.65 — 20.83 ± 0.47 —Butyrate 18.52 ± 4.92 16.89 ± 1.46 2.13 ± 0.598 — — 11.46 ± 1.46  20.6 ±3.96 — Cecal or Si contents SPF + A SPF + B SPF + Vanco SPF SI SPF SI +P μmol/g luminal contente Propionate — — 1.851 ± 1.41   2.69 ± 0.2412.08 ± 3.69 Acetate 53.95 ± 1.91 — 8.63 ± 1.73 15.19 ± 9.2  15.46 ±3.69 Butyrate — 29.78 ± 6.202  1.04 ± 0.903 7.183 ± 2.39  6.45 ± 2.11Select bacterial species ASF 356 (XIV) ASF 492 (XIV) Clostridium remosum(XVII) Clostridium bifermentans (XI) Bacteroides fragilisμmol/10{circumflex over ( )}5 CFU Propionate 62.39 ± 0.22  22.93 ± 0.10914.74 ± 0.526 1.147 ± 0.008 0.0517 ± 0.001 Acetate 220.0 ± 0.435 123.2 ±0.272 118.4 ± 0.526 1.973 ± 0.001  0.137 ± 0.001 Butyrate ND ND ND ND NDSCFA mix in mouse water bottle over time Input (Day 0) Day 1 Day 4 Day 7Day 10 Day 14 Day 90 mM Propionate 200 200 199.6 201.24 202.9 203.2 41.9Acetate 200 195 220.1 192.2 196.3 202.5 56.1 Butyrate 200 203.09 202.85202.27 202.69 202.19 38

To determine whether SCFA could directly affect cT_(reg) in a GFsetting, the present investigators isolated cT_(reg) from GF micetreated with propionate in vivo for three weeks and examined expressionof Foxp3 and interleukin 10 (IL-10), a key cytokine in T_(reg)-mediatedsuppression. The present investigators also isolated cT_(reg) from GFmice and stimulated them with propionate for 24 hours in vitro. Bothtreatments resulted in a significant increase in both Foxp3 and IL-10expression, as shown in FIGS. 1D and 1E. In vitro treatment increasedIL-10 production but not transforming growth factor-β (TGFβ) (FIGS. 1Dand 1E), a T_(reg)-mediated suppression factor, suggesting that SCFAspecifically induce Foxp3⁺ IL-10 producing T_(reg) subsets.

Exposure to the antibiotic vancomycin, which preferentially targetsGram-positive bacteria and disrupts the gut microbial community, reducedcT_(reg) to similar levels as those observed in GF mice, as shown inFIG. 1F. However, when SPF mice were treated with a combination ofvancomycin and SCFA, the reduction in cT_(reg) was completely restored(FIG. 1F).

Collectively, the foregoing results provide evidence that SCFA play acritical role in cT_(reg) homeostasis.

Example 2

Having determined that SCFA can restore cT_(reg) populations, furtherinvestigations were undertaken to determine whether SCFA could augmentcT_(reg) in SPF mice. As depicted in FIGS. 5A, 5B and 5C, treatment ofSPF mice with individual SCFA or a mixture of SCFA increased thefrequency and number of Foxp3⁺ cT_(reg) as well as Foxp3⁺IL-10⁺cT_(reg), and SCFA did not affect Foxp3⁺TGFβ⁺ cT_(reg) (FIG. 9). Changeswere not observed with SCFA treatment in small intestinal T_(reg)numbers, as illustrated in FIG. 10. To determine if SCFA effects have abroad impact on intestinal T cell populations, colonic Th17 and Th1cells were also examined, however no significant changes were evident(FIGS. 11 and 12). Furthermore, SCFA did not alter T_(reg) numbers inthe MLN (FIG. 13) or the spleen (FIG. 14) of SPF mice. Thus SCFAspecifically increase cT_(reg) numbers in healthy mice with aconventional microbiota.

These results may explain the benefits of dietary fibers and bacteria,such as clostridia and bifidobacteria that can increase colonic luminalSCFA production and modulate inflammation in mice and humans. Thepresent investigators measured SCFA production of species belonging toClostridium cluster XI (Clostridium bifermentans), XIV (ASF 356 and492), XVII (C. ramosum), and the bacteroides species, B. fragilis asClostridium cluster XIV members and B. fragilis affect cT_(regs). Asshown in Table 1, ASF 356 and 492 and C. ramosum generated morepropionate (14-62 vs 0.05-1.1 μmol/10⁵ CFU) and acetate (118-220 vs0.1-2 μmol/10⁵ CFU) as compared to the other strains.

A major mechanism by which T_(reg) regulate intestinal homeostasis andcontrol inflammation is through their ability to limit proliferation ofeffector CD4⁺ T cells (T_(eff)). The addition of SCFA to cT_(reg) andT_(eff) co-cultures increased the in vitro suppressive capacity ofcT_(reg), as shown in FIGS. 5D and 5E, indicating that these SCFAenhance not only cT_(reg) numbers but also their functional capacity.

In SPF mice, SCFA are taken up by colonic epithelial cells but alsodiffuse through the epithelial cell layer into the lamina propria wherethey can mediate their effects directly. To determine if SCFA directlyaffect cT_(reg), cT_(reg) were isolated from SCFA-treated SPF mice. Invivo treatment increased cT_(reg) Foxp3 and IL-10 expression (FIG. 6).cT_(reg) were also isolated from SPF mice and incubated with SCFA for 24hours in vitro. As illustrated in FIG. 5F, Foxp3 expression and IL-10expression and protein production all increased. Incubation with SCFAdid not alter cT_(reg) TGFβ levels (FIG. 5F).

As enhanced suppressive activity could be attributed not only to higherIL-10 levels per cT_(reg), but also to increased cT_(reg) proliferation,the present investigators examined cT_(reg) proliferation. Asillustrated in FIG. 7, cT_(reg) exhibited enhanced proliferation whencultured in the presence of propionate.

Taken together, the foregoing results indicate that SCFA may have abeneficial effect in healthy SPF mice and perhaps humans through theirability to directly increase Foxp3⁺IL-10 producing cT_(reg).

Example 3

In view of the findings that SCFA can directly influence cT_(reg),further investigations were performed to analyze the expression patternsof T_(reg) trafficking molecules. Although levels of the chemokinereceptor CCR9 or α4β7 integrin were not altered in propionate treated GFand SPF mice, levels of the cT_(reg) homing receptor GPR15 did increase,as illustrated in FIG. 8. Taken together, these data indicate that SCFAmay have a beneficial effect in SPF mice through their ability toincrease Foxp3+IL-10 producing cT_(reg) and cT_(reg) proliferativecapacity, as well as alter cT_(reg) GPR15 expression.

Considering that SCFA can influence cT_(reg) directly, furtherinvestigations were conducted to determine whether the observedinfluence was a receptor-mediated process. The G-coupled proteinreceptor 43 (GPR43), which is encoded by the Ffar2 gene, specificallybinds SCFA and through its expression on innate immune cells mediatesresolution of inflammatory responses (Maslowski et al., Nature 461,1282-1286 (2009); Nilsson, et al., Biochem. Biophys. Res. Commun. 303,1047-1052 (2003)). However, the role of Ffar2 and GPR43 in T_(reg) celldevelopment and function heretofore has not been probed.

GPR43 expression on T_(reg) isolated from secondary lymphoid tissues(spleen, MLN) was profiled and it was determined that intestinal T_(reg)and, particularly cT_(reg), had significantly higher levels of Ffar2than T_(reg) isolated from spleen or MLN. As depicted in FIG. 15A andFIG. 15B, on average cT_(reg) had higher levels of Ffar2. Thisexpression seemed dependent upon microbiota-derived signals asintestinal T_(reg) from GF mice had reduced GPR43 expression, on average4-fold lower for the GF colon (FIG. 15A). As a reference, cT_(reg) Ffar2expression levels were compared to colonic myeloid (CD11b⁺) cells, whichare known to express Ffar2 and, as shown in FIG. 15C, it was found thaton average CD11b⁺ cells expressed 1.6 fold more Ffar2 than cT_(reg).

To determine if Ffar2 contributes to cT_(reg) homeostasis and whetherGPR43 was responsible for cT_(reg) induction in SPF mice, furtherinvestigations were conducted by treating Ffar2^(−/−) mice andFfar2^(+/+) littermate control mice with propionate, which has thehighest affinity for Ffar2 (Le Poul et al., J. Biol. Chem. 278,25481-25489 (2003)), and cT_(reg) numbers were assessed. As illustratedin FIG. 15D, propionate significantly enhanced the percentage and numberof cT_(reg) in Ffar2^(+/+) control mice but had no effect in Ffar2^(−/−)mice, in which the levels of cT_(reg) were similar to those observed inthe water-treated control Ffar2^(+/+) mice. SCFA-mediated, enhancedcT_(reg) suppressive capacity was also dependent on Ffar2, as shown inFIG. 15H. The foregoing results indicate that Ffar2 is necessary forSCFA-mediated in vivo cT_(reg) induction.

To determine if GPR43 is required for direct stimulation by SCFA,cT_(reg) from Ffar2^(+/+) and Ffar2^(−/−) mice were isolated andincubated with propionate in vitro. Similar to SPF and GF cT_(reg),propionate enhanced both Foxp3 and IL-10 expression and IL-10 proteinlevels in Ffar2^(+/+) cT_(reg), but not in Ffar2^(−/−) cT_(reg), asillustrated in FIG. 15E. Furthermore, the present investigators examinedwhether propionate could restore cT_(reg) populations and numbers in thesetting of vancomycin treatment and, as illustrated in FIG. 15F, Ffar2was necessary. The foregoing therefore evidences that GPR43 is requiredfor complete induction of cT_(reg) by SCFA.

In view of the findings that SCFA can induce nT_(reg), furtherinvestigations were conducted to determine whether SCFA mediate theireffect through HDAC inhibition. GPR43^(+/+) and GPR43^(−/−) mice weretreated with propionate and HDAC expression was subsequently measured.The HDAC inhibitor trichostatin-A (TSA) increases T_(reg) geneexpression and suppressive capacity and HDAC6 and HDAC9 down-regulatenT_(reg) function. Given the findings presented herein that SCFA promotecT_(reg) homeostasis, it was hypothesized that SCFA mediate theireffects through HDAC inhibition. As illustrated in FIG. 15G, propionatetreatment of Ffar2^(+/+) mice reduced cT_(reg) expression of HDAC6 andHDAC9 (class IIb and IIa, respectively), but did not reduce expressionof the class I HDAC1 and HDAC2 or the class IIa HDAC7. Expression levelsin Ffar2^(−/−) were similar to untreated Ffar2^(+/+) mice. Western blotanalysis demonstrated that propionate treatment of cT_(reg) enhancedhistone acetylation, which also required the expression of Ffar2, asshown in FIG. 15I.

The foregoing results therefore indicate that SCFA may increase thenumbers and suppressive capacity of cT_(reg) through HDAC inhibitionthrough GPR43.

Example 4

Interactions between the gut microbiota and immune cells are criticalfor intestinal health and dysregulation of these interactionscontributes to inflammatory bowel disease (IBD). To test whether SCFAcan augment cT_(reg) function in vivo and ameliorate experimental IBD,further investigations were performed utilizing the T cell transfermodel of colitis. In this model, lymphopenic mice (e.g., RAG2^(−/−))were injected either with naïve T cells that results in severe colitis,or naïve T cells in combination with T_(reg) which reduces the severityof the colitis.

As shown in FIG. 17A, those mice receiving propionate or the SCFA mixwith naïve T cells and T_(reg) demonstrated less severe weight loss thanthose mice that received water alone (P<0.0001). Reduced severity ofcolitis in the presence of the propionate or the SCFA mix substantiatedthat these agents augmented the ability of T_(reg) to dampen intestinalinflammation (mean colitis score SCFA mix: 1.9±0.6P=0.0007, propionate:2.0±0.6P=0.0014 vs. water alone: 5.1±0.6). In contrast, as shown in FIG.16A those mice receiving only naïve T cells did not have reduced levelsof colitis (mean colitis score: 8.2±0.9 vs. T-reg+water alone 5.1±0.6,P=0.03). Representative H&E images are shown for the experimental groupsin FIG. 16B, with a 100 μm scale bar shown in the lower left of eachimage. As shown in FIG. 16B, the propionate and SCFA treated micedemonstrated reduced degrees of colonic crypt injury, inflammation, andhyperplasia compared to control mice.

As illustrated in FIGS. 17B and 17C, analysis of the cT_(reg)demonstrated an increase in the frequency and number of colonic LPFoxp3⁺ T_(reg) in those mice receiving propionate and the SCFA mix.Exposure of the mice to SCFA, however, did not result in conversion ofnaïve T cells to cT_(reg) (see, FIGS. 17B and 17C, comparing group 1 vs.group 2). To evaluate whether these effects were cT_(reg) intrinsic anddependent upon Ffar2, the present investigators performed the T celltransfer colitis model using RAG2^(−/−) recipients, wild-type naïve Tcells and Ffar^(+/+) or Ffar2^(−/−) T_(reg) with or without propionatein the drinking water. As illustrated in FIG. 17D and in FIG. 18, theeffect of propionate on intestinal inflammation was dependent upon Ffar2expression in T_(reg) as indicated by the colitis scores. As shown inFIGS. 17E and 17F, T_(reg) cell populations and cell numbers furthersubstantiated that the propionate effects on cT_(reg) were dependent onFfar2. The foregoing findings suggest that exposure to SCFA can augmentT_(reg) function, which was clinically significant and can ameliorateintestinal inflammation. Additionally, SCFA induced an accumulation ofFoxp3⁺ IL-10⁺ T_(reg) in the colon of GF and SPF mice, and oraladministration of SCFA protected against T cell-driven colitis throughenhanced T_(reg) cell function.

The foregoing studies demonstrate that short chain fatty acids (SCFA)are capable of inducing T_(reg) development in germ-free mice andprotect against colitis by enhancing T_(reg) population size andfunction in conventional mice via Ffar2 and GPR43. In the absence ofFfar2, SCFA failed to increase cT_(reg) expression of Foxp3 or thesuppressive capacity of cT_(reg). SCFA are potent HDAC inhibitors andtreatment of cT_(reg) with SCFAs led to increased histone acetylationand expression of Foxp3 and IL-10, an effect that required Ffar2. Thedata presented herein reveal a previously unidentified role for Ffar2and GPR43 linking the adaptive immune system, diet and the gutmicrobiota to intestinal homeostasis.

The incidence of IBD and other inflammatory diseases (e.g., autoimmunediseases) and obesity is steadily increasing in industrialized nations,and altered interactions between the gut microbiota and host immunesystem have been invoked as a cause (Lozupone, et al., Nature 489,220-230 (2012)). The western dietary pattern, specifically reducedingestion of plant-based fibers, may be a critical factor that links themicrobiome and disease (De Filippo et al., Proc. Natl. Acad. Sci. U.S.A.107, 14691-14696 (2010)). The findings disclosed herein affirm theconnectivities between diet, the gut microbiota and immune homeostasis.Clinically, such results provide evidence that supports the practice ofusing SCFA to treat patients with colonic inflammation (e.g., IBD,pouchitis, graft versus host disease) and support SCFA and/or dietaryinterventions that promote SCFA for the treatment of such colonicinflammatory diseases. Collectively, the data presented hereinsubstantiate that SCFA regulate intestinal immune responses, reduceinflammation and can restore intestinal homeostasis and thereby treatcolonic inflammation (e.g., inflammatory bowel diseases).

Materials and Methods:

Mice

Specified pathogen-free (SPF) mice were bred and housed in microisolatorcages in the barrier facility at the Harvard School of Public Health.Mouse studies and experiments were approved and carried out inaccordance with Harvard University's Standing Committee on Animals andthe National Institutes of Health guidelines for animal use and care.Foxp3^(YFP-Cre) mice on a C57BL/6 background were generously provided byDr. A. Rudensky (Memorial Sloan Kettering Cancer Center) (Turnbaugh etal., Nature 449, 804-810 (2007)). GPR43−/− mice were produced byDeltagen (Calif., USA) and heterozygous embryos provided by AstraZenecaTransgenic and Comparative Genomic R&D (Molndal, Sweden) (Hooper, etal., Science 336, 1268-1273 (2012)). GPR43 heterozygous cT_(reg) expresssimilar levels of GPR43 and Foxp3 as compared to GPR43 WT cT_(reg) byqPCR. GPR43 expression levels were examined in Foxp3⁻YFP⁻ and Foxp3⁻YFP⁺CD4+ T cells isolated from the colon, small intestine, mesenteric lymphnode, and spleen by RTqPCR (FIG. 20).

Germ-Free Mouse Experiments

Germ-free (GF) mice were bred and maintained in vinyl positive pressureisolators within the Germ-Free and Gnotobiotic core facilities at theHarvard Digestive Diseases Center at Brigham and Women's Hospital orChildren's Hospital Boston. GF experiments were performed at the Brighamand Women's Hospital Germ-Free and Gnotobiotic core facility. Mice weretreated for 21 days with either sodium acetate (150 mM), sodiumpropionate (150 mM), sodium butyrate (100 mM) or a SCFA mix (67.5 mMacetate, 40 mM Butyrate, 25.9 mM Propionate) in the drinking water. Inin vivo drinking water treatment experiments, control mice received pHand sodium-matched water. At the conclusion of the experiment mice wereremoved from isolators and processed immediately. Data on the stabilityof the SCFA mix solution are provided in Table 1.

SPF SCFA Intervention

For all SPF mouse experiments, mice were treated for 2 weeks with SCFAin the drinking water at the concentrations indicated above and inSmith, et al. Science 341: 1645 (2013). To compare oral versusintra-rectal administration of SCFA, mice were intra-rectally instilledwith either 200 μl of propionic acid (50 mM) or pH matched water, fortwo weeks, using an umbilical vein catheter 3.5 F. For T cell colitisexperiments, mice received either sodium propionate (150 mM) or the SCFAmix (described above) in the drinking water beginning at day 0.

Antibiotic Treatment

Mice were treated with vancomycin (500 mg/L; Sigma) in the drinkingwater for 4 weeks. Fluid intake was monitored and the antibioticsolution was changed every 3 days.

T Cell Transfer Model of Colitis

At Day 0, Naive CD4+ CD25⁻CD45RB^(hi) splenic T cells were FACS-sortedfrom BALB/c mice and injected i.p. into 6-8 week old BALB/c RAG2^(−/−)immunodeficient recipients (5×105 cells/mouse). At day 10, BALB/cRAG2^(−/−) immunodeficient recipients received CD4⁺CD25⁺CD45RB^(lo)splenic T_(reg) cells (75,000/mouse) isolated from BALB/c or C57BL/6mice Ffar2^(+/+) and Ffar2^(−/−) mice where indicated. Mice weremonitored weekly for weight loss and morbidity for 6-9 weeks as per theprotocol's experimental endpoint guidelines.

Lamina Propria (LP) Lymphocyte Isolation

To isolate LP lymphocytes, small and large intestines were collected andopened longitudinally and washed with PBS to remove fecal contents. Toremove epithelial cells, mucus and fat tissue, intestines were incubated2 times in EDTA (5 mM)/DTT (1 mM)/Dulbecco's PBS (calcium andmagnesium-free) solution (5 ml/colon) for 25 min at 37° C. Intestineswere then minced and collagenase-digested two times for 45 min at 37° C.in RPMI containing 1 mg/ml collagenase type VIII (SIGMA), 25 μg/ml DNaseI (Roche), 50 μg/ml Dispase (StemCell Technologies) and 0.01M HEPES(StemCell Technologies). The crude cell suspension was loaded onto a40%/90% Percoll (GE Healthcare) gradient and centrifuged at 720×g for 25min at room temperature with acceleration and brake turned off. LPlymphocytes were collected from the 40%/90% interface.

Flow Cytometry Staining

Intracellular staining of the transcription factors Foxp3, Helios andT-bet was performed using the Foxp3 Fix/Perm Buffer Set (Biolegend). Fordetection of intracellular cytokines, cells were first stimulated for 4h with 50 ng/ml PMA and 1 μg/ml ionomycin in the presence of Brefeldin A(All obtained from Sigma), followed by staining for surface markers.Cells were then fixed and permeabilized using the Foxp3 Fix/Perm BufferSet (Biolegend) and stained for intracellular cytokines. The followingantibodies were used: PE-labeled anti-Foxp3 (FJK-16s, eBioscience),Pacific Blue-labeled anti-Helios (22F6), PE-, FITC- or APC-labeledanti-CD4 (RM4-5), PECy7-labeled anti-CD3 (145-2C11), PE- or APC-labeledanti-IL-10 (JES5-16E3), PerCP-Cy5.5-labeled anti-IL-17 (TC11-18H10.1),APC- or PE-Cy7-labeled anti-IFNγ (XMG1.2), Pacific Blue-labeledanti-TGFβ1 (TW7-16B4), FITC-, PerCP-Cy5.5 or Pacific Blue-labeledanti-CD45 (30-F11), PerCP-Cy5.5 anti-CD45RB (C363-16A), PE- orFITC-labeled anti-CD25 (PC61), Brilliant Violet 421-labeled anti-CD127(A7R34), GPR43 rabbit polyclonal (Abcam Catalog #Ab118449, andanti-rabbit IgG DyLight649 (Poly4064). All antibodies were obtained fromBiolegend unless otherwise noted. Flow cytometry was performed usingLSRII and data were analyzed with FlowJo software (TreeStar, Inc.). Cellsorting was performed using a FACSAriallu at the Dana-Farber CancerInstitute Flow Cytometry Core.

SCFA Measurement

Cecal or small intestinal contents were collected immediately afteranimals were sacrificed and flash frozen in N₂(l). Samples were mashedin 500 μl HPLC grade water and centrifuged at >14,000×g, the resultingsupernatant was then passed through a 0.22 μm syringe filter to removebacterial cells and debris. Samples we then acidified with 1/10 volumeof 0.01M H₂SO₄, heated and passed through a condenser to isolatevolatile compounds within a sample. SCFA analyses were performed usingan Agilent 1200 series HPLC and a Poroshell 120 SB C18 column (2.7 μm,3.0×100 mm) with guard column (Agilent Technologies). Sulfuric acid(0.01M) was used as the mobile phase. SCFA concentrations were weightcorrected.

Single colonies were inoculated into chopped meat glucose media(Anaerobe Systems) and cultured in an anaerobic hood for 48 hrs at 37°C. Tubes were vortexed, 1 ml of media was transferred to an eppendorftube, and in parallel serial dilutions were grown on pre-reducedBrucella agar with 5% sheep blood to determine the colony forming unitsper ml of the liquid cultures. Eppendorf tubes were immediately removedfrom the anaerobic hood upon collection, spun at 14,000×g for 5 minutes,supernatant was transferred to a fresh tube, and samples were flashfrozen until they were processed as described above. A non-inoculatedchopped meat glucose media tube was used as a control and processed inthe same manner as the experimental samples. No propionate, acetate, orbutyrate were detected in the non-inoculated media tube. Butyrate wasnot detected in any of the culture supernatants and these data areconsistent with the short chain fatty profiles published for thesespecies in the Virginia Polytechnic Institute Anaerobe Laboratory ManualFourth Edition.

RNA Isolation

T_(reg) were isolated from the colon, small intestine, mesenteric lymphnode or spleen of C57BL/6 Foxp3^(YFP-Cre) mice and FACS-sorted based onexpression of YFP. RNA samples were prepared using the RNeasy Mini Kit(Qiagen) and cDNA was synthesized using the Bio-Rad iScript cDNASynthesis Kit. Real-time (RT)-qPCR was performed using the KAPA SYBRFAST Universal qPCR Kit (KAPA Biosystems) and a Stratagene MX3005P(Agilent Technologies). The following primer sequences were used:

Foxp3: (SEQ ID NO: 1) 5′-GGCAATAGTTCCTTCCCAGAGTT-3′ (SEQ ID NO: 2)5′-GGGTCGCATATTGTGGTACTTG-3′; IL-10: (SEQ ID NO: 3)5′-TTTGAATTCCCTGGGTGAGAA-3′ (SEQ ID NO: 4) 5′-GGAGAAATCGATGACAGCGC-3′;TGFβ1: (SEQ ID NO: 5) 5′-CCGCAACAACGCCATCTATG-3′ (SEQ ID NO: 6)5′-CCCGAATGTCTGACGTATTGAAG-3′; GPR43: (SEQ ID NO: 7)5′-AATTTCCTGGTGTGCTTTGG-3′ (SEQ ID NO: 8) 5′-ACCAGACCAACTTCTGGGTG-3′;HDAC1: (SEQ ID NO: 9) 5′-CCAAGTACCACAGCGATGAC-3′ (SEQ ID NO: 10)5′-TGGACAGTCCTCACCACG-3′; HDAC2: (SEQ ID NO: 11)5′-TGAAGGAGAAGGAGGTCGAA-3′ (SEQ ID NO: 12)5′-GGATTTATCTTCTTCCTTAACGTCTG-3′; HDAC7: (SEQ ID NO: 13)5′-CTCGGCTGAGGACCTAGAGA-3′ (SEQ ID NO: 14) 5′-CAGAGAAATGGAGCCTCTGC-3′;HDAC3: (SEQ ID NO: 15) 5′-CACCATGCCAAGAAGTTTGA-3′ (SEQ ID NO: 16)5′-CCCGAGGGTGGTACTTGAG-3′; HDAC6: (SEQ ID NO: 17)5′-CTGCATGGCATCGCTGGTA-3′ (SEQ ID NO: 18) 5′-GCATCAAAGCCAGTGAGATC-3′;HDAC9: (SEQ ID NO: 19) 5′-GCGGTCCAGGTTAAAACAGAA-3′ (SEQ ID NO: 20)5′-GCCACCTCAAACACTCGCTT-3′; GPR15: (SEQ ID NO: 21)5′-GGAGGACTGGCTCTTTCCTG-3′ (SEQ ID NO: 22) 5′-AAGGCTGGGTGCATGATAGC-3′.In Vitro T Cell Suppression Assay

CD4⁺CD25⁻ T effector cells (T_(eff)) were sorted from spleens of SPFmice and labeled with CFSE (Invitrogen) or Cell Trace Violet(Invitrogen) as per the manufacturer's instructions. T_(eff) cells wereplated at 5×10⁴ cells/well in 96-well round-bottom plates with 5×10⁴,CD4 depleted, irradiated (3000 rad) splenocytes, 1 μg/ml anti-CD3antibody and various numbers of CD4⁺CD25⁺ T_(reg) from the coloniclamina propria of SPF mice. Where indicated 0.1 mM sodium acetate,sodium propionate or sodium butyrate was added to the culture. After 96h, cells were collected and analyzed by flow cytometry.

In Vitro T Cell Stimulation

Colonic lamina propria T_(reg) were isolated from C57BL/6Foxp3^(YFP-Cre) mice as described above and FACS-sorted based onexpression of YFP. Isolated T_(reg) were cultured in RPMI 1640 mediumsupplemented with 10% FBS (Gibco), 4 mM L-glutamine, 80 U/ml penicillin,80 μg/ml streptomycin, 1 mM sodium pyruvate, 10 mM HEPES and 1×nonessential amino acids (all obtained from Cellgro). T_(reg) werestimulated with 1 μg/ml anti-CD3 antibody and with or without 0.1 mMsodium propionate for 24 h at 37° C. The division index is calculated bydividing the total number of cell divisions by the number of cells addedat the start of the culture and represents the average number ofdivisions a cell from the original population has undergone and includesthe undivided fraction in the calculation.

Western Blot

Colonic lamina propria T_(reg) were isolated and whole cell lysatesgenerated using RIPA buffer in the presence of protease inhibitors.Protein lysates were resolved using SDS-PAGE and transferred to PVDFmembrane using a Bio-Rad wet transfer apparatus. Blots were probed withantibodies directed against acetyl-histone H3 (K9) and histone H3 (Allfrom Cell Signaling Technologies). After incubation with the appropriateHRP-conjugated antibody, ECL was used for developing. Densitometryanalysis of Western blots was performed using Image-J software availableon the world wide web at rsbweb.nih.gov/ij/.

ELISAs

Cytokines were measured in culture supernatants using standardcytokines, antibodies and protocols. IL-10 was measured using the BDOptEIA ELISA Kit (BD Biosciences) and TGFβ1 levels were measured usingthe mouse TGF-beta 1 DuoSet (R&D Systems).

Histology and Colitis Scores

Colons were excised and cleaned with DPBS prior to fixation in 4% PFAand then processed by routine paraffin embedding, sectioning and H&Estaining. Colitis scores were determined by J.N.G., who was blinded tothe experimental parameters. Each of 4 histologic parameters were scoredas absent (0), mild (1), moderate (2), or severe (3): mononuclear cellinfiltration, polymorphonuclear cell infiltration, epithelialhyperplasia, and epithelial injury. The scores for the parameters weresummed to generate the cumulative histologic colitis score as previouslydescribed.

Statistical Analyses

GRAPHPAD PRISM Software was used for the calculation of statisticalmeasures, including mean values, standard errors, students' t test,Mann-Whitney test and Kruskal-Wallis test.

What is claimed is:
 1. A method of increasing the function of colonicregulatory T cells (cT_(reg)) in the gastrointestinal tract of asubject, wherein the cT_(reg) express a G-coupled protein receptor 43(GPR43), and wherein the method comprises contacting the cT_(reg) withan effective amount of a composition comprising one or more short chainfatty acids and thereby increasing the cT_(reg) function.
 2. A method oftreating colonic inflammation in a subject, wherein the method comprisesadministering an effective amount of a composition to the subject andthereby treating or preventing colonic inflammation, wherein thecomposition comprises one or more short chain fatty acids, wherein thecomposition increases immune function of colonic regulatory T cells(cT_(reg)) in the gastrointestinal tract of the subject, and wherein thecT_(reg) express a G-coupled protein receptor 43 (GPR43).
 3. A method ofreducing the incidence of relapse of inflammatory bowel diseases (IBD)in a subject, wherein the method comprises administering an effectiveamount of a composition to the subject and thereby reducing theincidence of relapse of IBD, wherein the composition comprises one ormore short chain fatty acids, wherein the composition modulates immuneresponse of the subject's colonic regulatory T cells _((CTreg)), andwherein the cT_(reg) express a G-coupled protein receptor 43 (GRP43). 4.The method of claim 2, wherein the one or more short chain fatty acidsinteract with the G-coupled protein receptor 43 (GPR43).
 5. The methodof claim 4, wherein the interaction of the one or more short chain fattyacids with the G-coupled protein receptor 43 (GPR43) inhibits histonedeacetylase (HDAC).
 6. The method of claim 5, wherein the HDAC is HDAC6.7. The method of claim 5, wherein the HDAC is HDAC9.
 8. The method ofclaim 2, wherein the one or more short chain fatty acids are selectedfrom the group consisting of formic acid, acetic acid, propionic acid,butyric acid, isobutyric acid, valeric acid, isovaleric acid andpharmaceutically acceptable salts thereof.